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STABLE KNOCK DOWN OF NON-ACCEPTABLE HLA MISMATCHES IN SOLID ORGAN TRANSPLANTATION.
Constanca Figueiredo, Axel Seltsam, Prof., Ute Holtkamp, Dr. and Rainer Blasczyk, Prof.. Hannover Germany, Hannover Medical School, 30625, Transfusion Medicine.

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HLA polymorphism represents the most relevant immunologic barrier in organ and stem cell transplantation. As RNA interference (RNAi) is capable of effectively knocking down specific transcripts, RNAi was used to selectively reduce cellular HLA class I expression. For this purpose, small interfering RNAs (siRNAs) were designed and chemically synthesised to target both β2-microglobulin (β2m) as well as the heavy chain transcripts. Sensitive sites in the target RNAs were identified using an in vitro translation system. The best siRNAs were used for knockdowns in B-LCL, K562 and HeLa cells. In order to achieve a stable reduction of HLA expression, lentiviral expression vectors were constructed encoding the sequences for short hairpin RNAs (shRNAs), which are subsequently processed intracellularly into functional siRNAs. Expression of HLA and β2m was determined by flow cytometry. Transfection of B-LCLs and K562 cells with siRNA targeting β2m resulted in a 50% suppression of β2m after 5 days. Using group- and gene-specific siRNAs targeting HLA-A transcripts, a reduction of up to 70% was observed after 6 days when performing repetitive transfections. When lentiviral vectors were used for transduction of shRNA, HLA-A suppressions of 70% in HeLa cells and 60% in B-LCLs were obtained, after 6 days without observing off-target effects. The present data strongly support the idea that knocking down HLA expression in class, gene and group-specific ways is extremely effective. The possibility to deliver the constructs by viral transduction, which offers both a stable transcript reduction and organ selective applicability, presents an exciting new immunotherapeutic approach in the field of organ and stem cell transplantation.